The asparagine-linked oligosaccharide chains of mammalian glycoproteins have been shown to be important determinats for the clearance of serum glycoproteins and for the sequestration of lysosomal hydrolases. Carbohydrate structures have also been implicated in a number of other biological functions. The final composition and structure of these oligosaccharides is determined by the concerted action of a series of glycosidases and glycosyltransferases that modify or "process" the protein-bound oligosaccharides. Mechanisms that regulate the final composition and structures of the oligosaccharides at specific glycosylation sites, however, have not yet been clarified. This application describes detailed investigations that will be carried out on some of the enzymes in the processing pathway, with major emphasis on a calcium activated Alpha-1,2-mannosidase and on GlcNAc transferase I from rabbit liver. The activities of these enzymes may play a key role in regulating whether the final structures of the oligosaccharide chains are of the complex or of the high-mannose type. The specific aims of the proposed research are to: 1) purify the Alpha-1, 2-mannosidase and GlcNAc transferase I, 2) characterize the physical and kinetic properties of the enzymes, 3) determine the mechanisms for enzyme substrate interactions, 4) reconstitute the enzymes in membranes composed of well defined phospholipids, 5) characterize the enzyme phospholipid interactions, and 6) investigate the effects of divalent cations and phospholipids on enzyme activity. Results from this research will provide significant new information about the properties of these enzymes in a lipid environment, and will provide insight into the mechanisms involved in controlling glycoprotein processing reactions.